**7. Modifications**

Genetic engineering is one of the methods for improvement of activity in hydrogen generation by microorganisms. Although the yields of generated hydrogen can be performed by optimization of the reaction conditions, genetic modifications seems to be the appropriate solution at the moment. The main idea of modification rely on implantation of other genes into the bacteria strains containing hydrogenase.

The *E. coli* are very frequently use in genetic modifications due to the well recognized metabolism of these bacteria. The *E. coli* are producing hydrogen as the result of

alcohols is observed. High concentration of VFA in the medium leads to the substrate inhibition (Kargi, 2010) as well as to the lowering of pH value (Liu, 2010) which consequently decreases the hydrogen yield or completely stops hydrogen generation. In hybrid systems photofermentation is the rate limiting step and slows down the overall reaction rate (Ozmihci, 2010). The unfavorable effect caused by the difference in the reactions rates can be counteracted by appropriate choice of concentrations of different strains of bacteria. The optimum concentration ratios can vary from 1:3.9 (Argun, 2010) even to 1:600 (Liu, 2010) depending on the strains of bacteria and types of substrates. The use of hardly soluble substrates such as e.g. starch leads towards formation of suspensions and flocculation of bacteria cells and further to limited accessibility of organic carbon to the bacteria and decrease in the yield of microbiologically generated hydrogen (Argun, 2009). The main advantage of one-step hybrid systems is the high rate and much higher yield of hydrogen produced in comparison to those obtained in the process of dark fermentation performed by one culture only. Further increase in the yield of hydrogen generated by hybrid systems can be achieved by application of two-step systems (Argun, Kapdan 2009).

The yield of hydrogen generation in the photofermentation process can be lowered by low access of light, inappropriate concentration of the medium, substrate inhibition, presence of ammonium ions or other contaminants (Ozmihci, 2010). Because a much greater number of parameters influence the yield of hydrogen in photofermentation than in dark fermentation, the former process should be performed in an independent photobioreactor. Application of two-step hybrid systems allows the use of wastes containing inhibitors of photofermentation process (e. g. ammonium ions) (Azbar, 2010). These inhibitors are neutral

Moreover, separation of these processes into two-step hybrid systems extends the list of organic substrates as it permits the use of highly thermophilic bacteria operating in temperatures higher than 70 °C (Ozgur, 2010). The natural organic substrates and wastes that can be used in two-step hybrid system. One mole of glucose theoretically generates four moles of hydrogen in dark fermentation, whereas acetic acid is the only side product (Antgenent, 2004). In practice, dark fermentation of liquid wastes generates much lower amounts of hydrogen (2.5-2.7 mole H2 per mole of glucose in waste) (Ueno, 1998, Yokoi, 2001, Yokoi, 2002). The hybrid systems are much more efficient. These results suggest that further development of two-step hybrid system can lead towards effective, economically

Genetic engineering is one of the methods for improvement of activity in hydrogen generation by microorganisms. Although the yields of generated hydrogen can be performed by optimization of the reaction conditions, genetic modifications seems to be the appropriate solution at the moment. The main idea of modification rely on implantation of

The *E. coli* are very frequently use in genetic modifications due to the well recognized metabolism of these bacteria. The *E. coli* are producing hydrogen as the result of

*Two-steps hybrid systems* 

for bacteria engaged in the dark fermentation.

other genes into the bacteria strains containing hydrogenase.

feasible commercial applications.

**7. Modifications** 

decomposition of formic acid in presence of formate-hydrogen liaze (FHL) representing the set of enzymes localized in the inner cell membrane. Hydrogenase 3 coded as *hycA* and formate dehydrogenase known as *fdhF* are the main components of the FHL. The presence of *hycA* gene limits the synthesis of *fhlA*, responsible for better activity of FHL towards hydrogen. Therefore the removal of hycA increases the *fhlA* gene expression and in consequence hydrogen production by 5-10%. (Hallenback, 2009). The research of the FHL genes expression were performed by Bisaillon *et al*. and other authors (Bisaillon, 2006, Turcot, 2008, Penfold, 2003) and they found almost two times higher rate of hydrogen generation for modified strain of E. coli HD701. Genes responsible for nickel-iron hydrogenases (hydrogenase I and II) coded by *hya* and *hyb* operons were found in the *E. coli* genom as well. It was found that elimination of these enzymes by genetic modification can result with almost 35% higher production of hydrogen (Hallenback, 2009, Bisaillon, 2006, Turcot, 2008). Other profits originating from genetic engineering are related to deactivation of enzymes responsible for transformations of glucose into lactic, succinic and fumaric acids. The removal of *ldhA* (lactic acid) and *frdBC* (succinic and fumaric acids) genes results in increase of hydrogen formation. The 1.4 fold higher amount of hydrogen were found by Yoshida *et al.* (Yoshida, 2006) in this situation. The new mutant strain of SR 15 can produce 1.82 mol H2/mol glucose what is close to the theoretical value (2 mol H2/mol glucose). Studies performed by *Maeda et al.* (Maeda, 2007) showed that bacteria BW2513 with seven modified genes (*hyaB, hybC, hycA,fdoG, frdC*, *ldhA and aceE*) generate 4.6 fold more hydrogen than wild-type strain.

The nitrogenase and uptake hydrogenase play an important role in the photofermentation process of hydrogen generation by PNS bacteria. The engineering of the mutants free of uptake hydrogenase is the basic task of gene modifications. Genes coding hydrogenase (*hup*) can be modified by resistance gene insertion into the *hup* genes or by deletion of *hup* genes (Kars, 2009, Kars, 2008, Kim, 2006). Appropriately modified *Rhodobacter spheroids* can generate hydrogen also in the absence of light (Kim, 2008).

Production of polyhydroxybutyrate (PHB) accompany hydrogen generation by PNS bacteria what applies the excess of reducing equivalents in other metabolic pathway. The PHB is the storage material stored in cytoplasm. This compound is formed in the environment rich in carbon compounds but lean in nitrogen (Kemavongse, 2007). The PHB is unwanted competition product accompanying hydrogen generation. The removal of genes responsible for formation of PHB syntase effectively stops generation of the polymer (Kim, 2006). Low activity in PHB formation not always results in an increase of hydrogen yield. Whereas in presence of lactate, malate or malate the amount of photogenerated hydrogen is not influenced by PHB (Hustede, 1993) the presence of acetate can increase photofermentation towards hydrogen. However, the importance of PHB as biodegradable polymer significantly increased in recent years. Therefore, simultaneous photogeneration of hydrogen and PHB gained economic dimension (Yigit, 1999).

There are genetic modifications influencing changes in the amount of LHC (light harvesting complexes). The reduction of pigment present in LHC diminish the self-shadow effect and therefore better access of light into deeper located cells. The decrease of amount of LH1 (Vasilyeva, 1999) complexes with maximum of absorption at 875 nm or those with absorption maximum at 800 and 850 nm (LH2) (Kim, 2006) can increase the amount of photo generated hydrogen. Genetic manipulations cannot lead to total elimination of the pigments (Kim, 2006).

Microbiological Methods of Hydrogen Generation 243

There are many electrons carriers such as cytochromes (proteins containing Fe) or ferredoxin. Moreover, the main enzyme in photofermentation - nitrogenase contains 24 atoms of iron in each molecule. The presence of iron ions in medium containing PNS bacteria is one of the very important factors influencing hydrogen productivity. At concentrations of Fe2+ ions lower than 2.4 mg/l there is no hydrogen in products. At concentrations higher than 3.2 mg/l the gradual decrease of evolved hydrogen is observed. It was assumed that non physiological coagulation of the cells can occurs (Zhu, 2007). Molybdenum is the second microelement playing an important role in photofermentaive hydrogeneration. The optimal concentration of molybdenum is 16.5 μmol/l (Kars, 2006).

The substrate yield in hydrogen production can be significantly improved by adding other strains of bacteria into the liquid medium. Improvement in photofermentation was achieved by adding halofilic archeons of *Halobacterium salinarum* type. The integral membrane protein - bacteriorhodopsin as the pump for the light excited electrons. The H+ ions are pumped out from cytoplasm outside the cell . The proton gradient is then engaged in ATP synthesis by *Rhodobacter sphaeroides* and this way increasing hydrogen generation. In this case, it is advised to use strains of PNS bacteria tolerating high concentrations of salts (Zabut, 2006) because of the high activity of bacteriorodopsyne in aqueous solution with high ionic

This work was supported by Polish Ministry of Science and Higher Education (grant no: N

Akkerman, I.; Janssen, M.; Rocha, J.; Wijffels, RH. (2002). Photobiological hydrogen

Alalayaha, WM.; Kalila, MS.; Kadhuma, AAH.; Jahima, JM.; Alaujb, NM. (2008). Hydrogen

Antgenent, LT.; Karmi, K.; Al-Dahhan, MH.; Wernn, BA.; Domiguez-Espinoza, R. (2004).

Argun, H.; Kargi, F. (2010). Bio-hydrogen production from Grodnu wheat starch by

Argun, H.; Kargi, F. (2009). Effects of the substrate and cell concentration on bio-hydrogen

Argun, H.; Kargi, F.; Kapdan, IK. (2009). Hydrogen production by combined dark and light

production: photochemical efficiency and bioreactor design, *Int J Hydrogen Energy*,

production using *Clostridium saccharoperbutylacetonicum* N1-4 (ATCC 13564), *Int J* 

Production of bioenergy and biochemicals from industrial and agricultural

continuous combined fermentation using annular-hybrid bioreactor, *Int J Hydrogen* 

production from ground wheat by combined dark and photo-fermentation, *Int J* 

fermentation of ground wheat solution, *Int J Hydrogen Energy*, vol.34, pp.4305-4311.

strength.

N204 185440)

**9. References** 

**8. Acknowledgement** 

vol.27, pp.1195-1208.

*Hydrogen Energy*, vol.33, pp.7392-96.

*Hydrogen Energy*, vol.34, pp.6181-6188.

*Energy*, vol.35, pp.6170-6178.

wastewater, *Trends In Biotechnol,* vol.22, pp.477-485.

The negative influence of ammonium ions on nitrogenase is well recognized. Therefore, genetic modifications of nonsensitive to NH4+ ions should be the subject for considerations. Among many methods reducing the role of ammonium ions in photofermentation is blockage of Calvin cycle via mutation of genes coding the RuBisCO enzyme. This way the excess electron stream is directed to nitrogenase even in the presence of NH4 + ions. Another modification can be achieved by disruption of proteins transporting NH4 + ions through cytoplasmic membrane. Strains of this type ( e.g. *Rhodobacter capsulatus*) loose their ability to regulate nitrogenase in presence of ammonium ions. (Qian, 1996). Such modifications allow to perform photofermentation even in the presence of molecular nitrogen. Although the amount of generated hydrogen is lower than in nitrogen free atmosphere but economically much more favorable (Yakunin, 2002).

Genetic modifications can be very effective but also troublesome and very expensive. Therefore other methods of process improvement are under investigations. Optimum value of pH equals 7. Photofermentation with *Rhodobacter sphaeroides* starting at pH=6.8 and ending at pH=7.5 results in significant drop of activity ( 7 times) but PHB concentration is tripled (Jamil, 2009).

Photofermentative bacteria belongs to mesophilic microorganisms and operate between 30 and 35 oC. Therefore, any critical temperatures act against high yield of hydrogen. For example *Rhodobacter capsulatus* operating at temperatures varying from 15-40 oC produce 50% less hydrogen than the same bacteria kept at constant temperature of 30 oC (Özgüra, 2010).

The access of photobacteria to the light with appropriate length and intensity play a crucial role m hydrogen photogeneration. Better access of light induce better phosphorylation and in consequence more effective synthesis of ATP and better yield of photofermentation (Kars, 2010).

Although the PNS bacteria absorb light in wide spectrum 400-950 nm the range of 750-950 nm is the most important (Eroglu, 2009, Ko, 2002). The light intensity is as well important as their wavelength. For *Rhodobacter sphaeroides* the amount of generated hydrogen grows linearly from 270 W/m2 (4klx) to 600 W/m2 (~ 10 klx). Below 270 W/m2 no activity of bacteria is observed (Miyake, 1999, Uyar, 2007).

Application of illumination with wavelength longer than 900 nm results in overheating of the system. This require additional cooling systems because of decrease the amount of generated hydrogen. An application of appropriate filters cutting the unwanted range of spectrum seems to be the only solution in this situation (Ko, 2002). Considering natural irradiation one should remember about day-night periodicity. It was found, however, that amount of generated hydrogen is even higher under periodic irradiation than under the continuous one (Eroglu, 2010, Koku, 2003). The day-night illumination induces better activity of nitrogenase what results from better adjustment of PNS bacteria to live in natural conditions (Meyer, 1978).

The presence of organic compounds, also those containing nitrogen (except NH4 + ions) is the key issue for the photofermentation. However, presence of macro and microelements at appropriate concentration can influence the hydrogen productivity. Iron belongs to the most important ones. This element exists mainly as the cofactor of proteins engaged in metabolism. Process of photofermentation, strictly related to the transport of electrons.

The negative influence of ammonium ions on nitrogenase is well recognized. Therefore, genetic modifications of nonsensitive to NH4+ ions should be the subject for considerations. Among many methods reducing the role of ammonium ions in photofermentation is blockage of Calvin cycle via mutation of genes coding the RuBisCO enzyme. This way the

modification can be achieved by disruption of proteins transporting NH4+ ions through cytoplasmic membrane. Strains of this type ( e.g. *Rhodobacter capsulatus*) loose their ability to regulate nitrogenase in presence of ammonium ions. (Qian, 1996). Such modifications allow to perform photofermentation even in the presence of molecular nitrogen. Although the amount of generated hydrogen is lower than in nitrogen free atmosphere but economically

Genetic modifications can be very effective but also troublesome and very expensive. Therefore other methods of process improvement are under investigations. Optimum value of pH equals 7. Photofermentation with *Rhodobacter sphaeroides* starting at pH=6.8 and ending at pH=7.5 results in significant drop of activity ( 7 times) but PHB concentration is

Photofermentative bacteria belongs to mesophilic microorganisms and operate between 30 and 35 oC. Therefore, any critical temperatures act against high yield of hydrogen. For example *Rhodobacter capsulatus* operating at temperatures varying from 15-40 oC produce 50% less

The access of photobacteria to the light with appropriate length and intensity play a crucial role m hydrogen photogeneration. Better access of light induce better phosphorylation and in consequence more effective synthesis of ATP and better yield of photofermentation (Kars,

Although the PNS bacteria absorb light in wide spectrum 400-950 nm the range of 750-950 nm is the most important (Eroglu, 2009, Ko, 2002). The light intensity is as well important as their wavelength. For *Rhodobacter sphaeroides* the amount of generated hydrogen grows linearly from 270 W/m2 (4klx) to 600 W/m2 (~ 10 klx). Below 270 W/m2 no activity of

Application of illumination with wavelength longer than 900 nm results in overheating of the system. This require additional cooling systems because of decrease the amount of generated hydrogen. An application of appropriate filters cutting the unwanted range of spectrum seems to be the only solution in this situation (Ko, 2002). Considering natural irradiation one should remember about day-night periodicity. It was found, however, that amount of generated hydrogen is even higher under periodic irradiation than under the continuous one (Eroglu, 2010, Koku, 2003). The day-night illumination induces better activity of nitrogenase what results from better adjustment of PNS bacteria to live in natural

The presence of organic compounds, also those containing nitrogen (except NH4+ ions) is the key issue for the photofermentation. However, presence of macro and microelements at appropriate concentration can influence the hydrogen productivity. Iron belongs to the most important ones. This element exists mainly as the cofactor of proteins engaged in metabolism. Process of photofermentation, strictly related to the transport of electrons.

hydrogen than the same bacteria kept at constant temperature of 30 oC (Özgüra, 2010).

+ ions. Another

excess electron stream is directed to nitrogenase even in the presence of NH4

much more favorable (Yakunin, 2002).

bacteria is observed (Miyake, 1999, Uyar, 2007).

tripled (Jamil, 2009).

conditions (Meyer, 1978).

2010).

There are many electrons carriers such as cytochromes (proteins containing Fe) or ferredoxin. Moreover, the main enzyme in photofermentation - nitrogenase contains 24 atoms of iron in each molecule. The presence of iron ions in medium containing PNS bacteria is one of the very important factors influencing hydrogen productivity. At concentrations of Fe2+ ions lower than 2.4 mg/l there is no hydrogen in products. At concentrations higher than 3.2 mg/l the gradual decrease of evolved hydrogen is observed. It was assumed that non physiological coagulation of the cells can occurs (Zhu, 2007). Molybdenum is the second microelement playing an important role in photofermentaive hydrogeneration. The optimal concentration of molybdenum is 16.5 μmol/l (Kars, 2006).

The substrate yield in hydrogen production can be significantly improved by adding other strains of bacteria into the liquid medium. Improvement in photofermentation was achieved by adding halofilic archeons of *Halobacterium salinarum* type. The integral membrane protein - bacteriorhodopsin as the pump for the light excited electrons. The H+ ions are pumped out from cytoplasm outside the cell . The proton gradient is then engaged in ATP synthesis by *Rhodobacter sphaeroides* and this way increasing hydrogen generation. In this case, it is advised to use strains of PNS bacteria tolerating high concentrations of salts (Zabut, 2006) because of the high activity of bacteriorodopsyne in aqueous solution with high ionic strength.
